Chimeric proteins with phosphatidylserine binding domains

ABSTRACT

Chimeric proteins comprising soluble Tissue Factor (sTF) and another subunit (e.g., annexin V) are described. The proteins promote blood clotting and/or inhibit cancer by targeting sTF to specific receptors such as phosphatidyl serine (PS) on activated cells. These chimeric proteins are useful in treating patients with excessive bleeding due to inborn problems, drug therapy, trauma or surgery and/or as an anti-cancer therapy, for example by causing blood vessels feeding cancers to become clotted, thereby preventing adequate flow of blood to a tumor, which in turn will lead to tumor inhibition and death or may be used in a therapy to cause clotting within blood vessels that pose a threat in the subject in non-cancerous conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Application Ser. No. 60/659,938, filed Mar. 9, 2005, theentirety of which is hereby expressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

Hemostasis (the process of initiating blood coagulation in order to stopbleeding) may be abnormal and possibly life-threatening under a numberof circumstances, yet there is a need for improved methods of inducinghemostasis under special conditions. Hemostasis is initiated byplatelets, which are blood cells that cause occlusion of holes in bloodvessels. In the course of doing so, platelets become “activated”. Oneaspect of their activation is the translocation of phosphatidyl serine(PS) from the inner half of their plasma membrane to the outer half.Once this occurs, certain blood coagulation proteins bind to the surfaceof the platelet and interact in the process known as “bloodcoagulation”. The end result of that process is the formation of theenzyme thrombin (also called Factor IIa). Thrombin has several importantroles. First, it converts the blood protein fibrinogen into fibrin. Eachfibrin binds to several other fibrin molecules, forming a fibrin clot,which supports the platelets that are attempting to stop bleeding.Second, thrombin also activates platelets, increasing the numberinvolved in the hemostatic process. And third, thrombin affects otherplasma proteins, such as Factor XI, in a manner that accelerates thebiochemistry of blood coagulation. The first proteins that interact onthe surface of platelets (or other cells with exposed PS) are TissueFactor (TF), the activated form of Factor VII (called Factor VIIa), andFactor X.

Thrombin generation is initiated by the interaction of the plasma serineprotease, Factor VIIa, with its protein cofactor, TF. TF is amembrane-bound protein not expressed on the surface of cells in contactwith the bloodstream until they become activated. Upon its expression,TF binds either Factor VII (promoting its activation to Factor VIIa), orFactor VIIa, increasing its catalytic efficiency in converting Factor Xto Factor Xa. Expression of the extracellular domain of TF (amino acids1-219, or a 3-219 residue portion of the entire protein) in E. coligenerates a polypeptide of about 26 kDa that retains the ability to bindto Factor VIIa and to allosterically activate it. This truncated TF(called soluble TF or sTF) does not bind to cellular membranes and istherefore generally much less efficient than native TF in promotingFactor VII autoactivation or activation of Factor X by Factor VIIa.Engineering of the cDNA encoding sTF so that it was expressed on thesurface of mammalian cells as a glycosylphosphatidylinositol-anchoredprotein resulted in a protein with the same specific procoagulantactivity as native TF, underscoring the importance of membraneattachment for this protein.

It has long been recognized that congenital Factor VIII deficiency ischaracterized by abnormal thrombin generation when blood coagulation istriggered by low concentrations of TF. More recently recognized is thefact that disorders of platelet function are also associated withdecreased thrombin generation. The vitamin K-dependent blood coagulationproteins, which are required for thrombin generation, assemble on thesurface of activated platelets, endothelial cells, and/or monocytes bybinding to anionic phospholipids, especially PS. Thrombin, a potentplatelet agonist, amplifies the activation of platelets initiated bycontact with sub-endothelial collagen exposure. Delayed thrombingeneration may therefore underlie or amplify the bleeding tendencyaccompanying disorders of plasma coagulation factors, or bloodplatelets.

Others have reported that the intravenous injection of thromboplastin(membrane-bound TF) into animals results in generalized activation ofthe coagulation, as does injection of both sTF and Factor VIIa,resulting in beneficial effects upon bleeding in experimental animals,suggesting that sTF might serve as the basis of a therapy designed toreduce bleeding.

A therapeutic protein effective in enhancing hemostasis and coagulationin a subject in need thereof would be desirable.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Protein purification and western blotting of recombinantproteins. Top, SDS-PAGE and Coomassie blue staining. Middle, Westernblot using a mouse monoclonal antibody to human TF. Bottom, Western blotusing mouse monoclonal antibody to His₆. M, molecule weight marker;Lanes 1-5 are identical in panels A, B, and C: lane 1, sTF-annexin V(sTF-annV); lane 2, sTFM-annexin V (sTFM-annV); lane 3, annexin V; lane4, sTF; lane 5, recombinant TF. Lane 6, panel A: bovine serum albumin;panel B: recombinant TF (Innovin®, Dade Behring, Newark, Del.).

FIG. 2. Influence of recombinant proteins on plasma coagulation. A, Thecoagulation time of citrated human plasma with different concentrationsof native TF (▪), sTF (▾), or sTF-annV (●). The total amount of PC/PSadded to each sample was 10 mM (PC:PS molar ratio, 4:1). Shown aremeans±SEM. B, Plasma coagulation times with sTF (▾), annexin V (▴), bothsTF and annexin V (♦), or sTF-annV (●) measured using a dilute partialthromboplastin time protocol, as described in Experimental Procedures.C, Plasma coagulation times, in the absence of added phospholipids, withdifferent concentrations of sTF (▾), annexin V (▴), sTF-annV (●), orsTFM-annV (∘). Shown are means±SEM.

FIG. 3. Phospholipid binding of sTF-annV, and sTFM-annV. The ability ofsTF-annV (●) and sTFM-annV (∘) to bind PC/PS vesicles was compared tothat of annexin V (▴) by assessing the ability of each protein todisplace FITC-annexin V from a phospholipid suspension, as described inExperimental Procedures. The curves were computer using non-linearregression and a one site ligand binding equation. Data are expressed asthe percent (mean±SEM) of EDTA-elutable fluorescence.

FIG. 4. Autoactivation of Factor VIIa in the presence of native TF,sTF-annV or sTF. Factor VII (80 nM) was added to equimolarconcentrations of native TF (▪), sTF (▾), or sTF-annV (●) in thepresence of PC/PS (810 mM, PC:PS molar ratio=4:1) and CaCl₂. At theindicated times, aliquots were removed and the reaction stopped bydilution in EDTA-containing solutions. The Factor VIIa content of eachsample was then determined by measuring the rate of hydrolysis ofChromozym t-PA in the presence of added sTF and CaCl₂. Shown aremeans±SEM.

FIG. 5. Generation of Factor Xa by Factor VIIa in the presence ofrecombinant proteins. A, Various concentrations of Factor X were addedto 1 nM Factor VIIa, 5 mM PC/PS (molar ratio, 4:1), 5mM CaCl₂ andChromozym X substrate in the presence of 5 pM native TF (▪), sTF (▾),sTF-annV (●), or sTFAA-annV (∘). Initial rates of substrate hydrolysiswere measured as described in Experimental Procedures, to which theMichaelis-Menten equation was fit. B, Initial rates of Factor Xageneration were measured in solutions containing 28 nM Factor X, 5 mMCaCl₂ 1 nM Factor VIIa, 0.347 mM PC/PS and various concentrations of sTF(▾) or sTF-annV (●).

FIG. 6. The effect of sTF-annV or Factor VIIa on the aPTT ofheparin-treated plasma. A, aPTT assays of plasma containing heparinsodium (1 unit/mL) was measured in the presence of variousconcentrations of Factor VIIa (●) or sTF-annV (▪). B, The aPTT of plasmacontaining enoxaparin sodium (1 unit/mL) was measured in the presence ofvarious concentrations of Factor VIIa (●) or sTF-annV (▪). The dottedlines show the mean clotting time for the aPTT assay in the absence ofheparin or enoxaparin. Shown are means±SEM. (Most error bars are toosmall to be seen.)

FIG. 7. The effect of sTF-annV on the tail bleeding time. Panel A: Mice(n=13) were given a subcutaneous injection of enoxaparin sodium (20mg/kg), and two h later they were anesthetized and the tail bleedingtime determined as described in the Experimental Procedures. Immediatelyupon the cessation of bleeding, the animals were injected intravenouslywith sTF-annv (90 mcg/kg) in TBS. Five min later the bleeding time wasmeasured again. The differences were significant at a level of p=0.01(paired two-tailed t-test). Panel B: Tail bleeding times were measuredas described above. 2 hours after receiving SQ enoxaparin (or saline)animals were injected intravenously with sTF-annV (90 mcg/kg) orequivalent molar amounts of sTF, annexin V or both sTF and annexin V.Tail bleeding times were measured 10 min later. sTF-annV shortened thebleeding time of mice that did not receive enoxaparin (p>0.2) as well asthose that did (p<0.0001, two-tailed unpaired t-test with Welch'scorrection). Two animals were studied in each of the groups that did notreceive enoxaparin, and 4 in each of the enoxaparin-treated groups.

DETAILED DESCRIPTION OF THE INVENTION

The present invention contemplates delivering chimeric proteinscomprising soluble Tissue Factor (sTF), or effective portions thereofhaving activity sufficient to enhance anidolytic activity of Factor VIIaby at least 10-fold, to sites of vascular injury where they function asa hemostatic agent while minimizing the chance of inducing disseminatedintravascular coagulation (DIC). Although targeting might be undertakenby using an antibody specific for activated platelets, endothelial cellsor monocytes, a targeting moiety capable of binding to all three celltypes would be preferable, insofar as it would be less sensitive toalterations in the number of targeted cells that might be present in anygiven individual. Since each of the cell types of interest expressesphosphatidylserine (PS) on its surface when activated, the presentinvention contemplates a sTF chimeric protein (e.g., sTF-annV) for usein targeting sTF to PS-containing membranes wherein the sTF is coupledto a PS-binding domain such as annexin V (AnnV), a human PS-bindingprotein which has been shown to bind to activated platelets, endothelialcells and monocytes. Arguing against the use of annexin V in this mannerwas the fact that it is an anticoagulant protein, due to its ability tocompete with vitamin K-dependent proteins for binding to PS-containingmembranes.

In a preferred embodiment of the invention, sTF comprises 217 aminoacids (SEQ ID NO:2) of the human TF attached to the amino terminus ofAnnV, (it may also be attached to the carboxy terminus, and may belinked to the amino terminus or carboxy terminus via a linker sequence).As noted before, TF is normally bound to cell membranes because itcontains an intramembranous anchoring region, However, sTF, which lacksthe anchoring region and was cloned and expressed over 10 years ago, isapproximately 1000-fold less active than the native protein (TF). sTF isencoded for example by a cDNA comprising SEQ ID NO:1.

Soluble Tissue Factor (sTF) as contemplated herein comprises SEQ IDNO:2, or any subsequence thereof having a cysteine in position 207 andwhich retains activity sufficient to enhance the amidolytic activity ofFactor VIIa by at least 10 fold. Thus the sTF contemplated for useherein may comprise a truncated portion of sTF comprising amino acids1-207, 5-207, 10-207, 15-207, 20-207, 25-207, 30-207, 35-207, 40-207,45-207, 50-207, 55-207, 60-207, 65-207, 70-207, 75-207, 80-207, 85-207,90-207, 95-207, 100-207, or 105-207 and all sequences inclusive between1-207 and 105-207, including for example 2-207 and 104-207. Theinvention further comprises any alternative cDNA which encodes SEQ IDNO:2 or a functionally active truncated portion thereof as describedherein, for example the alternative cDNA which encodes SEQ ID NO:2 maycomprise a cDNA similar to SEQ ID NO:1 but which has conservativelysubstituted codons (i.e., a coding having a different nucleotidesequence but which encodes the same amino acid).

The invention further comprises any of the sTF sequences or truncatedsequences as described herein which further comprises an additionalser-gly sequence or just a gly residue on the N-terminal portion of SEQID NO:2, thereby providing a 219, or 218 amino acid sequence,respectively, and wherein the cysteine residue at position 207 of SEQ IDNO:2 is thus shifted to position 209, or 208, respectively.

The second domain of the sTF-AnnV chimeric protein is comprised of theentire human protein, Annexin V (SEQ ID NO:4) or of an effectivePS-binding portion thereof. AnnV is a 35 kDa protein encoded for exampleby SEQ ID NO:3 and is produced in many cells and binds to certainphospholipids, notably phosphatidylserine as noted above. PS is normallyfound on the inner half of the plasma membrane (of all cells). Enzymaticmechanisms exist that can translocate PS to the outer half of the plasmamembrane, where it may be bound by AnnV in the presence of calcium.

A linker region (such as, but not limited to, SEQ ID NO:6, as encoded bythe cDNA having SEQ ID NO:5) is optionally present in the chimericprotein between the sTF domain and the AnnV domain. In anotherembodiment, the chimeric protein of the present invention may also havea non-functional tag sequence at the carboxy and/or amino terminus, orinternally, and which primarily comprises for example 6-histidines (SEQID NO:8, as encoded by a cDNA having SEQ ID NO:7), which is used tobenefit purification of the chimeric protein, and which preferably isexcised therefrom prior to use of the chimeric protein in therapeuticadministration.

In an alternate embodiment, a mutant form of sTF is used in the chimericprotein. In one embodiment, the sTF mutant has alanine residues atpositions 163 and 164 rather than the lysine residues found at thosepositions in the native sTF (or in positions 165 and 166 of the sTFhaving 219 amino acids, for example). This alternate embodiment isreferred to herein as “sTFAA-AnnV” (SEQ ID NO:10, encoded for example bya cDNA having SEQ ID NO:9). In two other embodiments, sTF mutants haveglutamic acid residues or glutamine residues as substitutions at the 163and 164 positions referred to herein as “sTFEE” (SEQ ID NO:12 as encodedfor example by a cDNA having SEQ ID NO:11) and “sTFQQ” (SEQ ID NO:14 asencoded for example by a cDNA having SEQ ID NO:13, respectively). Eachof these mutants or the original sTF, may include (but are not limitedto) any or all of the following substitutions: ala at position 13, 131,163, 164 or 183, asn at position 42 or 138, trp at position 48, ser atposition 52, asp at position 128, glu at position 163 or 164, or gin atposition 129, 163 or 164.

In fact, the present invention contemplates that at any of thesepositions, non-polar amino acids may be substituted with polar aminoacids, or other non-polar amino acids, polar amino acids may be replacedwith non-polar amino acids of other polar amino acids, positivelycharged amino acids may be replaced with negatively charged, polar ornon-polar amino acids, or other positively charged amino acids, andnegatively charged amino acids may be replaced with positively charged,polar or non-polar amino acids, or other negatively charged amino acids.

The chimeric protein sTF-annV of the present invention thereforepreferably comprises, for example a protein comprising SEQ ID NO:2 or aneffective portion thereof linked with a protein comprising SEQ ID NO:4or an effective portion thereof. The sTF-annV protein may furthercomprise a linker, including but not limited to, a linker having SEQ IDNO:6 positioned between the sTF and annV sequences. The sTF-annVprotein, with or without the linker sequence, may comprise an internalor external tag sequence such as SEQ ID NO:8 for aiding in purificationof the sTF-annV protein. Any other effective linker sequence and/orpurification tag sequence may be used in lieu of SEQ ID NO:6 and/or SEQID NO:8.

In some embodiments of the invention it may be desirable for thechimeric protein to have a shortened serum half-live to minimize theserum residence time to limit the potential for the chimeric protein tocause unwanted thrombosis, or so that the chimeric protein is eliminatedafter a particular amount of thrombin has been formed. In theseembodiments of the invention, a cleavage site, such as a plasmincleavage site or thrombin cleavage site can be positioned between thesTF and annV sequences of any of the chimeric proteins described herein.For example, a plasmin cleavage site having SEQ ID NO:16 (as encoded forexample by a cNDN having SEQ ID NO:15), or a thrombin cleavage site suchas SEQ ID NO:18 (as encoded for example, by a cDNA having SEQ ID NO:17)can be inserted at a position between the sTF and annv sequences (withor without the linker sequences and/or purification tag sequence).

To extend the serum half-life of increase their activity, the inventionalso contemplates chimeric proteins containing multiple sTF domains,linked to the amino or carboxy terminus of Annexin V (such as theprotein having SEQ ID NO:19 as encoded for example by a cDNA having SEQID NO:20).

The invention also contemplates chimeric proteins comprising sTF, ormultiple sTF domains (or their derivatives as described herein) andother non-AnnV PS binding domains of proteins such as synpatotagmin I,or other proteins known to those skilled in the art, which would serveto anchor the sTF domain(s) to PS-containing membranes.

Examples of other PS-binding proteins that can be used in substitutionfor AnnV include, but are not limited to, Annexin family members,lactadherin, domains found in proteins known to bind PS, such as FactorV/Va, Factor X/Xa, Factor II/IIa, Factor VII/VIIa, Factor IX/IXa, FactorVIII/VIIIa, Spectrin, Class B Scavenger receptor type I, Protein KinaseC, and proteins containing the C2 domains of protein kinase C (thisincludes synaptotagmins), Rabphilin family members, the PS receptor,endothelial lectin-like OxLDL receptor-1 (LOX-1), antibodies to PS,phosphatidylserine decarboxylase, MARCKS (myristoylated, alanine-richprotein kinase C substrate), PS-p68, Myosin, Erythrocyte protein 4.1,hemoglobin, Calponin family members, S100A, S100B, calcyclin-bindingprotein family members, milk membrane-glycoprotein, MFG-E8 (milk fatglobule-EGF factor 8), and other PS-binding motifs known to those ofordinary skill in the art.

In one embodiment of the invention, prior to administration of thesTF-AnnV (or any other chimeric protein contemplated herein), a subjecthaving a tumor is first given an injection of chemotherapy which causeschanges in the tumor or tumor blood vessels causing an increased bindingof sTF-AnnV, for example by increasing PS expression on the surfaces oftumor or blood vessel cells. Administration of the sTF-AnnV wouldfollow, and would bind to the tumor and tumor blood vessel in enhancedamounts. Chemotherapy is defined as any treatment that causes death ofcancer cells, (i.e., drugs, chemicals, hormone, vitamins, etc.) as iswell known by those of ordinary skill in the art. Another approach is toadminister radiation therapy to the area (alone or with a drug therapy)in order to enhance the efficacy and/or binding of the sTF-AnnV.

Cancer cells receive signals from their environment that allow them togrow, travel, and invade normal tissues. They differ from normal cellsin that many have increased numbers of certain “receptor” molecules ontheir surface, which help the cells survive. This invention contemplatesin one embodiment new proteins designed to block tumor cell surfacereceptors including tissue factor and the urokinase receptor therebyaltering tumor cell behavior.

In one embodiment, the invention contemplates chimeric proteins designedto inhibit signaling through cellular receptors important for cancercells, and possibly normal inflammatory cells. In particular theinvention contemplates (1) novel proteins that bind to the urokinasereceptor, thereby preventing the binding of the two proteins thatnormally result in signaling through that receptor (urokinase andplasminogen activator inhibitor-1, also called PAI-1, and (2) novelproteins that bind to cell surfaces and contain the extracellular domainof the cell surface protein, tissue factor, which serves as a receptorfor the plasma protein called Factor VIIa. Since the complex comprisedof (native) tissue factor and Factor VIIa are able to generate signalsthat affect cancer cell behavior in a manner that is dependent upon thecytoplasmic tail of tissue factor, the chimeric proteins contemplatedherein that comprise sTF will act as a sink for Factor VIIa, divertingit from the tissue factor on the surface of the cancer cell. Theinvention also contemplates novel proteins that bind both to theurokinase receptor and to Factor VIIa, thereby keeping cancer cells frombenefitting from their own cell surface tissue factor and urokinasereceptor molecules. The invention also contemplates mutants of thetissue factor domains and/or the urokinase receptor domains to increasethe affinity of each of these domains for their ligands.

The urokinase receptor and tissue factor are also found on macrophagesof the so-called chronic inflammatory cells. The chimeric proteinsdescribed herein can also be useful in treating human or animal diseasesin which ongoing macrophage-mediated inflammation plays a role inunremitting disease or illness.

In another embodiment, the invention comprises sTF linked to ATF(sTF-ATF), a chimeric protein which blocks both the TF and urokinasereceptors of the cell. ATF is the amino-terminal protein fragment ofurokinase (amino acids 1-135 of the urokinase A chain, thereby denotedATF). ATF (SEQ ID NO:22, encoded by a cDNA having SEQ ID NO:21 forexample) binds to the urokinase receptor but, unlike full lengthurokinase, is not internalized.

The chimeric proteins of the present invention may comprise, in lieu ofAnnV, one or more peptides which occupy receptors of tumor vasculatureand other organs including homing peptides as described in U.S. Pat.Nos. 6,576,239; 6,491,894; 6,528,481; 6,610,651; and 6,933,281, each ofwhich is hereby expressly incorporated herein by reference.

Other peptides that bind to the tumor vasculature and which may comprisethe protein of the present invention include but are not limited to:HWGF (SEQ ID NO:23)-motif peptides (selective inhibitors of matrixmetalloproteinase-2 and matrix metalloproteinase-9, also known asgelatinase A and gelatinase B), for example the synthetic peptideCTTHWGFTLC (SEQ ID NO:24) targets angiogenic blood vessels, inhibits themigration of human endothelial cells and tumor cells, and also preventstumor growth and invasion in animal models and improves survival of micebearing human tumors.

As noted above, any of the tumor treatment methods contemplated hereinmay include an initial step of performing a scan with Annexin V (notsTF-Annexin V) in order to identify subjects who would be expected tohave a normal or enhanced response to treatment. A preferred subject isone who demonstrates uptake of annexin V into a tumor, without excessiveuptake in non-tumorous areas. This would be a more efficacious treatmentbecause some potential subjects might have enhanced uptake withoutantecedent drug and/or radiation therapy (and thus wouldn't necessarilybenefit from these ancillary treatments with their attendant sideeffects), while others might not. The latter would be more likely tobenefit by first getting chemotherapy or radiation treatment, therebyallowing for a synergistic interaction between the sTF-AnnV and the“priming” therapy.

In an annexin scan (performed before sTF-AnnV administration), Annexin Vsticks to activated platelets caught up in blood clots. Therefore, anAnnexin V scan might identify those subjects having an increased riskfor binding sTF-AnnV and who may experience pathological thrombi innon-tumorous vascular beds (i.e., to suffer a stroke, heart attack, deepvenous thrombosis, etc.). By doing an Annexin V scan first, the safetyprofile of the user may be improved by avoiding those at greater riskfor this complication. Annexin V scans can be performed by a variety ofimaging techniques (specifically magnetic resonance imaging in additionto nuclear medicine imaging), allowing for the development of newimaging compounds. Alternatively, subjects may be given an intravenousinjection of annexin V, prior to administration of sTF-annV (with orwithout chemotherapy and/or radiotherapy), in order to prevent bindingof sTF-annexin to blood vessels wherein thrombosis would be unwanted.

Experimental Procedures

Materials

Recombinant membrane-anchored TF, recombinant sTF (TF₂₋₂₁₉), andmonoclonal antibodies to Factor VII and TF were prepared as described. Amonoclonal antibody reacting with the His₆ tag of recombinant proteinswas from Cell Signaling Technology (Beverly, Mass.). Chromozym t-PA(N-methysulfonyl-D-phenylalanylglycyl-L-arginine-4-nitroanilide acetate)and Chromozym X(N-methoxycarbonyl-D-norleucyl-glycyl-L-arginine-4-nitranilide acetate)were from Roche Diagnostics Corp (Indianapolis, Ind.). Purified humanFactors X, Xa, VII, and VIIa were from Enzyme Research Laboratories(South Bend, Ind.). Unless otherwise specified, all other regents werefrom Sigma Chemical Co.

Phospholipids were obtained from Avanti Polar Lipid (Alabaster, Ala.) asstock solutions in chloroform and used to prepare unilamellarphospholipid vesicles. Aliquots were mixed at a molar ratio ofphosphatidylcholine (PC) to PS of 4:1, and the chloroform removed byevaporation under argon. Phospholipids were resuspended in 0.1 M NaCl,0.05 M Tris-HCl, pH 7.5, and sonicated until the suspension was almostclear, yielding PC/PS vesicles. The phospholipid concentration wasdetermined by phosphate analysis.

Relipidation of TF into phospholipid vesicles was performed using theoctyl-beta-D-glucopyranoside method, typically at TF:phospholipid molarratios of 1:8700. The effective TF concentration was determined bytitrating with increasing concentrations of Factor VIIa in solutionscontaining Chromozym t-PA and comparing the resulting amidolyticactivity to that obtained by incubating Factor VIIa with knownconcentrations of sTF. The phospholipid concentration of the TFpreparation was determined by phosphate analysis so that comparableamounts of PS were present when TF was compared directly to sTF-annV.

Fluorescein isothiocyanate (FITC)-annexin V was prepared by incubatingFITC with Annexin V at a 2:1 molar ratio for 1 hr in the dark at roomtemperature in 0.5 M carbonate buffer, pH 9.5. Unbound FITC was removedby gel filtration in 150 mM NaCl, 0.01M Tris-HCl, pH 8.2, followed bydialysis against the same buffer for an additional 48 hr.

Construction of Expression Vectors

Annexin V cDNA (in the pET-22b(+) vector) was a generous gift from Dr.Antony Rosen. The sTF-annV construct was generated by ligating the cDNAencoding amino acids 2-219 of TF to the 5′ end of the annexin V cDNA.The sTF cDNA was cloned by PCR using the forward primer5′-CATGCCATGGCAGGCGCTTCAGGCACTACAAATAC-3′ (SEQ ID NO:25), and thereverse primer 5′-CCCAAGTCTTGGGTTCTCTGAATTCCCCTTTCTC-3′ (SEQ ID NO:26).A linker sequence encoding (GGGGS)₃ was inserted between the sTF andannexin V cDNA using QuikChange® Multi Site-Directed Mutagenesis Kit(Stratagene, La Jolla, Calif.) with the following primer:5-GGGGMTTCAGAGAAGGTGGCGGTTCAGGCGGTGGAGGTTCAGGAGGTGGCGGATCMTGGCACAGGTTCTC-3′ (SEQ ID NO:27). The sTF-annV gene was cloned intothe NcoI and HindIII sites of the pET-22b(+) vector. This vector codesfor a protein with a His₆-tag at the carboxy terminus. A chimeracomposed of a mutated form of sTF linked to annexin V (sTFM-annV) wascreated by site-directed mutagenesis. Residues 164 and 165 of sTF (i.e.,residues 165 and 166 of the 219 amino acid sTF) were mutated with theQuikChange® Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla,Calif.) from lysine to alanine using the primer5′-CTTTATTATTGGAAATCTTCMGTTCAGGAGCCGCMCAGCCAAAAC AAACACTMTGAGTTTTTG-3′(SEQ ID NO:28). The sTFM-annV gene was cloned into the NcoI and HindIIIsites of pET-22b(+). DNA sequencing confirmed the composition of allthree constructs.

Expression and Purification of sTF-annV, sTFAA-annV and Annexin V.

The pET-22b(+) vectors with the sTF-annV, sTFAA-annV, or annV genes wereinserted into E. coli strain BL-21/DE3 (Novagen, Madison, Wis.). Thisvector provides a signal peptide that directs the recombinant protein tothe periplasmic space. Expression was induced when the A₆₀₀ of thebacterial suspension reached 0.8 by adding 0.3 mMisopropyl-beta-D-thiogalactopyranoside (IPTG) for 15 hr at 25° C. Cellswere harvested, subjected to osmotic shock (5 mM MgSO₄), and theperiplasmic fraction was collected. Recombinant proteins were purifiedby immobilized metal affinity chromatography using a His-Select column(Sigma, St. Louis, Mo.) equilibrated with 0.3 M NaCl, 0.05 M NaH₂PO₄, pH8.0. The column was washed with the same solution in the presence of 10and 20 mM imidazole and then eluted with 250 mM imidazole. The eluatewas dialyzed against 0.1 M NaCl, 50 mM Tris-HCl pH 7.4. The purity andidentity of recombinant proteins were examined by SDS-PAGE and Westernblotting.

Phospholipid Binding Assay

The affinity of chimeras for phospholipid vesicles containing PS wasdetermined by modifications of published assays using a commerciallyavailable aPTT coagulation reagent (Dade®Actin® FSL Dade Behring,Newark, Del.) as the source of PS. The reagent was reconstitutedaccording to the manufacturer's instructions and diluted 1:10 with 140mM NaCl, 0.01M HEPES, pH 7.5 (HBS) plus 2.5 mM Ca⁺⁺ (HBS-Ca⁺⁺). Thediluted phospholipid suspension (10 mL) was incubated with 50 nMFITC-annexin V (a concentration resulting in saturation of the PSpresent in the assay, as determined experimentally) in HBS-Ca⁺⁺ incoated microcentrifuge tubes (Slickseal, National Scientific, Claremont,Calif.). After incubation at 37° C. for 15 min, the mixtures werecentrifuged at 16,000×g for 20 min. The pellets were washed in HBS-Ca⁺⁺and then resuspended in HBS-Ca⁺⁺ containing various amount of each ofthe unlabeled competing proteins (annexin V, sTF-annV or sTFAA-annV).After an additional 12 hr incubation at 37° C., the mixtures werecentrifuged and the pellets washed in HBS-Ca⁺⁺. The pellets were thenresuspended in HBS plus 10 mM EDTA, and incubated for 16 hr at roomtemperature to elute bound FITC-annexin V. The tubes were centrifugedand the supernatant fluid removed. The amount of FITC-annexin in thesupernatant was determined by measuring its fluorescence (excitationwavelength, 485 nm; emission wavelength, 535 nm) in a fluorescentmicroplate reader (Victor2, Wallac, PerkinElmer, Boston, Mass.).

Factor VII Autoactivation Assay

The ability of the chimeric proteins to promote the autoactivation ofFactor VII was assessed by adding each (final concentration, 80 nM) toFactor VII (final concentration, 80 nM) in 0.1 M NaCl, 50 mM Tris-HCl,0.1% bovine serum albumin, pH 7.4 (TBS) with 5 mM CaCl₂ (TBS/Ca²⁺). Forexperiments utilizing sTF-annV or sTF, PC/PS vesicles were added to theincubation mixtures at a composition and concentration identical to thatpresent in the native TF preparation (total phospholipid concentration,810 mM, PC:PS molar ratio=4:1). At selected intervals, 10 mL aliquotswere removed from each incubation mixture and transferred to polystyrenetubes containing 40 mL TBS plus 5 mM EDTA to stop the reaction. Theamount of Factor VIIa generated was assayed by adding 150 mL ofTBS/Ca²⁺, excess sTF, and Chromozym t-PA (final concentrations, 5 mM,124 nM and 5 mM, respectively) and measuring the change in A₄₀₅.

Factor VIIa Binding Assay

The sTF/TF-induced increase in Factor VIIa amidolytic activity was usedto quantify the binding of these proteins to Factor VIIa. Increasingconcentrations of sTF-annV, sTFM-annV or sTF were incubated with FactorVIIa (5 nM) in TBS/Ca²⁺ in 96 well assay plates. After 10 min at roomtemperature, Chromozym t-PA was added (1 mM final) and the initial rateof substrate hydrolysis was measured at 405 nm using a microplate reader(Molecular Devices, Menlo Park, Calif.). The background activity ofFactor VIIa in the absence of sTF, sTF-annV or sTFM-annV was subtractedfrom the measured values. Kinetic parameters were calculated using Prism4 (GraphPad Software, San Diego, Calif.).

Factor X Activation

The activation of Factor X by Factor VIIa in the presence of sTF,sTF-annV, sTFAA-annV or native relipidated TF was monitored in acontinuous one stage assay. sTF, sTF-annV, sTFM-annV or native TF wereadded to 1 nM Factor VIIa, Factor X, PC/PS (molar ratio, 4:1) and 0.5 mMChromozym X in TBS/Ca²⁺. The rate of chromogenic substrate hydrolysis(change in A₄₀₅) was monitored over 20 min and converted to Factor Xaconcentrations by reference to a standard curve prepared with purifiedhuman Factor Xa. The derivative of the resulting parabolic progresscurve was taken (Softmax Pro, Molecular Devices, Menlo Park, Calif.) todetermine the rate of Factor Xa generation and kinetic parameters werecalculated using Prism 4 (GraphPad Software, San Diego, Calif.).

Plasma Coagulation Assays

Coagulation Times

Clotting times of normal human citrated plasma following the addition ofCaCl₂ in the presence of various concentrations of recombinant proteinsand PC/PS (molar ratio, 4:1) were measured with a mechanicalcoagulometer (ST-4, Diagnostica Stago, Parsipanny, N.J.), which had anupper limit of measurement of 999 s.

Dilute Activated Partial Thromboplastin Time

A commercially available aPTT reagent (Dade® Actin®, Dade Behring) wasdiluted 1:50 in TBS and used to measure the coagulation time of citratedhuman plasma in the presence of various concentrations of sTF, annexin Vor sTF-annV. Plasma was incubated with dilute aPTT reagent andrecombinant proteins for 3 min at 37° C., followed by the addition of 25mM CaCl₂. The time required for a clot to form was measured with amechanical coagulometer.

Activated Partial Thromboplastin Time (aPTT)

The aPTT of plasma containing either 1 unit/mL heparin sodium (AmericanPharmaceutical Partners, Inc. Los Angeles, Calif.) or enoxaparin sodium(Lovenox®, Aventis Pharmaceuticals, Bridgewater, N.J.) and variousconcentrations of sTF-annV or Factor VIIa was measured with acommercially available aPTT reagent (Actin® FSL; Dade Behring, Newark,Del.) according to the manufacturer's instructions.

Mouse Tail Bleeding Times

Mice were housed in accordance with and studied using a protocolapproved by the University of Oklahoma Health Sciences CenterInstitutional Animal Care and Use Committee. They were injectedsubcutaneously with enoxaparin sodium and two hours later wereanesthetized with pentobarbital (60 mg/kg, given i.p.). The tail waswarmed in normal saline at 37° C., transected with a scalpel blade at apoint where it was 2 mm in diameter, and then placed in a tube of normalsaline maintained at 37° C. The time required for bleeding to stop wasrecorded. In preliminary dose-finding experiments, increasing doses ofenoxaparin sodium were administered. Once a suitable dose was identified(20 mg/kg), a second bleeding time was performed 5 min after the first.The additional bleeding time was performed by transecting the tail at apoint 5mm proximal to the first. The second bleeding times were notsignificantly different than the first (p=0.3, two-tailed, pairedt-test, Prism 4, Graphpad Software). Untested animals were then treatedin a similar manner, but received an intravenous injection of sTF-annV(90 mcg/kg) immediately after the first bleeding time determination. Asecond bleeding time was performed 5 min after the injection. Atwo-tailed, paired t-test was used to compare the two bleeding times. Anadditional group of animals were then studied. Two hours after the SQinjection of saline or enoxaparin (20 mg/kg), mice were given IVinjections of sTF-annV (90 mcg/kg), saline, sTF, annexin V or acombination of sTF and annexin V. All proteins were injected in amountsequimolar with sTF-annV. Tail bleeding times were measured 10 minuteslater.

Results

Expression and Purification of Annexin V and Annexin V Chimeras

cDNA constructs encoding three proteins (annexin V, sTF-annV andsTFM-annV) were expressed in the periplasmic space of E. coli and theproteins were purified by immobilized metal affinity chromatography (SeeFIG. 1 for analyses of the purified proteins by SDS-PAGE and westernblotting).

sTF-annV Accelerates Plasma Coagulation

In initial studies, the abilities of sTF, native TF, and sTF-annV toaccelerate plasma coagulation in the presence of identicalconcentrations of added phospholipid and CaCl₂ were compared. As shownin FIG. 2A, sTF-annV and native TF shortened the plasma coagulation timeto a much greater extent than did sTF. A dilute aPTT assay was then usedto determine whether the effects of sTF-annV could be duplicated byadding sTF to annexin V. Plasma was recalcified in the presence ofdilute aPTT reagent (used as a source of phospholipid) and sTF, annexinV, the combination of sTF and annexin V, or sTF-annV. As shown in FIG.2B, the simultaneous addition of sTF and annexin V did not reproduce theprocoagulant effect of sTF-annV. Under these conditions, sTF-annVdemonstrated a biphasic effect on plasma coagulation, prolongingcoagulation at higher concentrations.

These results suggested that at lower concentrations the procoagulanteffect was due to the sTF domain, while at higher concentrations theanticoagulant effect of annexin V predominated. To test this hypothesis,a construct was prepared in which the lysines at positions 165 and 166of the TF/sTF domain (164 and 165 of the 218 aa sTF) were mutated toalanine, a change known to impair Factor X activation. The resultingchimera (STFM-annV) also exhibited biphasic effects on plasmacoagulation (FIG. 2C), but had less procoagulant activity than sTF-annVand exhibited its anticoagulant activity at lower concentrations. Giventhe evidence that sTF-annV and sTFM-annV were able to alter plasmacoagulation, detailed functional characterization of their constituentdomains was undertaken.

Phospholipid Binding Activity

The ability of sTF-annV and sTFM-annV to bind PS was assessed with amodification of a published PS-binding assay in which annexin V,sTF-annV or sTFM-annV competed with FITC-annexin V for binding tophospholipid vesicles. As shown in FIG. 3, both chimeras exhibitedPS-binding activity comparable to annexin V. The K_(d) for annexin V was12 nM; for sTF-annV, 13 nM, and sTFM-annV, 11 nM.

Factor VII Autoactivation Activity

It has been shown previously that TF, but not sTF, promotesautoactivation of Factor VII in the presence of PS and Ca²⁺. The failureof sTF to accelerate Factor VII autoactivation has been attributed tothe low affinity of sTF:VIIa complexes for PS-containing membranes.Since the annexin V domain of the chimera exhibited high affinity forPS-containing liposomes, we postulated that sTF-annV would promoteFactor VII autoactivation in a manner comparable to TF, rather than sTF.As shown in FIG. 4, sTF-annV promoted Factor VII autoactivation at arate comparable to native TF, rather than sTF (which was inactive).These results also suggest that sTF-annV is able to bind Factor VII aswell as native TF.

Factor VIIa Binding Activity

To measure the binding of Factor VIIa to the chimeras, we quantified theincrease in Factor VIIa's amidolytic activity when its binds to TF/sTF.Because sTF does not bind membranes well, we performed the analysis inthe absence of PS. Both sTF-annV and sTFM-annV increased the amidolyticactivity of Factor VIIa to the same extent as sTF, indicating that theannexin V domain did not interfere with the ability of the sTF domain tobind Factor VIIa (data not shown). The K_(d) for binding of Factor VIIato sTF was 8.17 nM; for sTF-annV, 7.05 nM; and for sTFM-annV, 3.47 nM,all in the absence of phospholipid vesicles.

Factor X Activation

The effect of sTF-annV and sTFM-annV on the rate of Factor X activationby Factor VIIa was assessed. As shown in FIG. 5A, the rate of Factor Xactivation in the presence of either sTF-annV or sTFM-annV was greaterthan that seen in the presence of sTF. As expected from prior studies ofsTFM, sTF-annV was more potent than sTFM-annV in supporting Factor Xactivation. As shown in Table 1, the catalytic efficiency(k_(cat)/K_(m)) of Factor VIIa in the presence of sTF-annV wasapproximately 67% of that found in the presence of native TF, butapproximately 5-fold greater than that of sTFAA-annV.

Since high concentrations of sTF-annV were associated with prolongationof the plasma coagulation times, we studied the effect of increasingconcentrations of sTF-annV on the rate of Factor X activation by FactorVIIa. As shown in FIG. 5B, sTF-annV exhibited a biphasic effect onFactor X activation, paralleling its effects on plasma coagulation.TABLE 1 V_(max) k_(m) k_(cat) k_(cat)/k_(m) Cofactor nM · min⁻¹ nM s⁻¹μM⁻¹s⁻¹ TF  0.71 ± 0.008 46 ± 1 2.37 51.5 sTF-annV 0.66 ± 0.03 64 ± 72.2 34.4 sTFAA-annV 0.71 ± 0.06 389 ± 58 2.37 6.1Effect of sTF, sTF-annexin V, and sTFAA-annV on the Rate of Factor XActivation by Factor VIIaEffect of sTF-Annexin on Coagulation in the Presence of Heparin

Heparin is a commonly used anticoagulant that works by enhancing theability of the serpin, antithrombin, to inhibit (primarily) Factor Xaand thrombin. We added unfractionated heparin sodium or enoxaparin, alow molecular weight heparin, to citrated plasma along with variousconcentrations of sTF-annV and then determined the plasma aPTT. sTF-annVshortened the aPTT of plasma containing either 1 unit/mL unfractionatedheparin (FIG. 6A) or 1 unit/mL enoxaparin (FIG. 6B). Factor VIIa, usedas a therapeutic agent to treat bleeding in numerous clinical conditionsassociated with decreased thrombin generation, also decreased the aPTTof heparin-treated plasma, although less potently than sTF-annV (FIGS.6A and 6B).

Effect of sTF-annV on Mouse Tail Bleeding Times

Although the template bleeding time assay in humans is thought topredominantly reflect platelet function, defects in thrombin generationin mice are reflected by prolonged bleeding times. We thereforeadministered enoxaparin to normal mice to determine whether sTF-annVwould affect an in vivo measure of thrombin generation. Mice wereinjected subcutaneously with enoxaparin sodium (20 mg/kg), a drug knownto inhibit thrombin generation and the bleeding time measured 2 hourslater. After the bleeding from the bleeding time wound stopped, theanimals were given an intravenous injection of sTF-annV (90 mcg/kg) andthe bleeding time repeated 5 min later. (Preliminary experiments showedthat a second bleeding time in enoxaparin-treated animals was notsignificantly different than the first, p=0.3, paired two-tailedt-test.) As shown in FIG. 7A, sTF-annV significantly shortened thebleeding time of enoxaparin-treated mice (p=0.01, paired two-tailedt-test). Enoxaparin treatment resulted in a mean bleeding time of 10.7minutes (median, 6.1 minutes). The mean bleeding time after sTF-annVtreatment was 1.4 minutes (mean, 1.1 minutes). The mean bleeding time ofuntreated animals was 1.2 minutes.

The tail bleeding time was measured in additional groups of mice.sTF-annV shortened the bleeding time of animals not treated withenoxaparin (1.5±0.7 min versus 3.6±0.2 min, p>0.02, unpaired t-test withWelch's correction.; FIG. 7B). Naïve animals were then treated withenoxaparin (20 mg/kg, SQ) 2 h before they were injected IV with sTF-annV(90 mcg/kg) or equimolar amounts of sTF, annexin V, or the combinationof sTF and annexin V. As shown in FIG. 7B, shortening of the bleedingtime was only seen in animals treated with sTF-annV (p<0.0001,two-tailed unpaired t-test with Welch's correction), showing that thechimera itself, and not its individual constituents, was responsible forthe shortening of the bleeding time.

Previous studies have shown that the isolated extracellular domain of TF(sTF) retains a conformation sufficient to allow binding to Factor VIIa,and enhancement of its cleavage of a small tripeptidyl syntheticsubstrate. sTF is much less efficient in promoting the activation ofFactor X by Factor VIIa, however. This appears to be due to relativelylow affinity of sTF and the sTF-VIIa complex for membrane surfaces. Bysubstituting a glycosylphosphatidylinositol anchor for the TFtransmembrane domain, Paborsky et al. were able to create amembrane-bound sTF variant that had full procoagulant activity,indicating the ability of sTF to function as well as native TF ifappropriately tethered to a cell surface.

Others have coupled sTF to peptide moieties and demonstrated activationof the coagulation system in vitro and in vivo. Each utilized atargeting domain specific for a particular cell type or anatomic regionand all of these studies were directed towards developing anti-canceragents, rather than novel hemostatic compounds.

In the present work, a chimeric protein containing the sTF domain hasbeen created in a preferred embodiment to have several novel features:(1) it is “poly-specific” with regard to the cell types that play a rolein hemostasis; and (2) it has a membrane-targeting domain that hasanticoagulant properties. Thus, although annexin V's potency as aPS-binding protein makes it attractive as a targeting moiety, itsanticoagulant action cautions against its use in a construct designed tobe a procoagulant hemostatic agent.

The initial studies of sTF-annV were therefore directed towardsdetermining whether it was in fact procoagulant, and if so, how itsprocoagulant activity compared to that of both sTF and native TF.Because we recognized that the ratio of annexin V to phospholipid couldbe important, we utilized phospholipid suspensions, rather thanactivated cells, to maintain consistency, during our analysis. Once wefound that sTF-annV was procoagulant (FIG. 2A), we established that theprocoagulant activity was specific to the chimera, and could not bereproduced by adding equimolar amounts of a mixture of independent sTFand annexin V (FIG. 2B). We also prepared a chimera (referred to hereinas sTFM-annV) in which amino acids 165 and 166 of sTF were mutated fromlysine to alanine, a change known to significantly reduce theprocoagulant activity of TF and found that it had diminishedprocoagulant activity (FIG. 2C). Finally, we explored the consequencesof altering the ratio of the chimera to the PS present in the reactionmixture. We found that for both sTF-annV and sTFM-annV, an increase inthe protein:PS ratio beyond a certain point resulted in an anticoagulanteffect (FIG. 2C). Thus, targeting of sTF to a membrane surface by theannexin V domain is procoagulant when there is a functional excess ofPS-bearing sites that can bind other coagulation factors. As the amountof annexin V present increases, those sites become less accessible tocoagulation Factors VII/VIIa, X, VIII, IX, V and II, and coagulationslows.

We then conducted analyses of the functional domains comprisingsTF-annV, to determine if linking them together caused a loss or gain offunction of either. Tests of the chimera's ability to bind PS-containingvesicles (FIG. 3), promote Factor VII autoactivation (FIG. 4), enhanceFactor VIIa's amidolytic and Factor X-cleaving activity (FIG. 5)indicated that both the sTF and annexin V domains were functional,although native TF promoted the catalytic efficiency of Factor VIIatowards Factor X twice as well as did sTF-annV (see Table 1).

These studies indicated that sTF-annV could function as a procoagulant,but its procoagulant activity was heavily dependent upon thephospholipid concentration. Since the local concentration of PS at awound site in vivo is not precisely known, in vitro experiments havelimited ability to predict the behavior of a PS binding protein (such assTF-annV) in vivo. In order to test sTF-annV in vivo under conditionswhere the PS concentration would be determined by the body's response toinjury, we performed tail bleeding times in mice, a method known to besensitive to defective thrombin generation in the setting of the prioradministration of enoxaparin, a low molecular weight heparin, used toimpair thrombin generation. In preparation for this experiment, westudied the effect of sTF-annV on plasma coagulation in the presence ofenoxaparin, as well as unfractionated heparin, and contrasted it withthe effects of Factor VIIa, a protein currently used to treat patientswith a variety of bleeding disorders. Both Factor VIIa and sTF-annVshortened the coagulation time of heparin- and enoxaparin-treatedplasma, although sTF-annV was several orders of magnitude more potentthan Factor VIIa (FIG. 6).

We treated mice with increasing doses of enoxaparin, establishing a dosethat increased two sequential bleeding times reproducibly. We theninjected sTF-annV by intravenous tail vein injection between the firstand second tail bleeding time. In all animals tested, the secondbleeding time was significantly shorter than the first. Using additionalanimals, we found that the effect of sTF-annV could not be reproduced byindependently injecting sTF, annexin V or a mixture of the two. Becausehuman Factor VIIa is not fully active in mouse plasma, it could not beused for direct comparisons. The dose of sTF-annV was chosen because itis equivalent (on a weight basis) to a commonly recommended recombinantFactor VIIa dose.

These studies demonstrate that seemingly antagonistic protein domainsmay be combined in a chimeric molecule to generate a protein whoseactivity is not simply determined by the net effect of its dominantdomain. The protein sTF-annV exhibits an effect that is reflective ofeither of its opposing domains, depending upon the conditions. Theability of sTF-annV to exhibit both procoagulant and anticoagulantactivity makes it a unique among hemostatic molecules.

Because AnnV binds to PS, and PS is required for blood coagulation, AnnVwhen used alone prevents proteins of the clotting mechanism from bindingto PS thus AnnV actually inhibits blood coagulation and thus generallywould not be considered to be a hemostatic agent. Even though sTF, inhigh concentrations can accelerate blood coagulation, a complex of sTFand AnnV, as described in this invention, would not be an obvious methodof improving blood coagulation, and might well be expected by thoseknowledgeable in the art to either have no hemostatic effect or to havean anticoagulant effect due to the presence of the AnnV domain.

The current invention has proven to be more effective than sTF alone, atlow concentrations. Without wishing to be constrained by theory, thismay be due to the fact that the AnnV domain brings sTF close to themembrane surface, where Factor X is bound.

Plasma-derived hemostatic agents have been shown to induce thrombosis(unwanted blood coagulation) in some patients and thrombosis has alsobeen seen in some trauma patients treated with recombinant Factor VIIa.

The present invention has a means (the AnnV domain or other domainsdescribed herein) of localizing sTF to areas of activated platelets,which is likely to provide a greater measure of safety, as compared tocomparably effective hemostatic compounds which circulate throughout thevasculature.

Our work has shown that at higher concentrations, the sTF-AnnV proteinloses its procoagulant (hemostatic) effectiveness, and becomes ananticoagulant, as evidenced by a prolongation of the blood clottingtime. This is likely due to the fact that at high concentrations, theeffect of the AnnV domain becomes more important than its effect oflocalizing the sTF domain to the membrane surface due to occupation ofpotential binding sites for Factor X. No other hemostatic agent isreported to have the property of being anti-coagulant at highconcentrations. This feature is likely to make sTF-AnnV a saferhemostatic agent than others currently available.

Utility

The chimeric proteins of the present invention can be used in a varietyof therapeutic applications related to hemostasis and thrombosis.

Use as a Hemostatic Agent.

The present invention contemplates the administration of the chimericprotein sTF-AnnV (or sTF chimera linked to other PS binding elements asdescribed herein) to bleeding patients with the intent of increasing thegeneration of thrombin in the bleeding area, leading to a cessation ofbleeding. Often, applying pressure or a suture will cause bleeding tostop. However, there are some circumstances when this cannot be easilyaccomplished wherein a compound such as sTF-AnnV would be useful. Suchsituations include (1) bleeding in the presence of an anticoagulantdrug, (2) bleeding due to the lack (congenital or non-congenital) of oneor more coagulation factors such as may be seen in trauma and massivetransfusion, vitamin K deficiency, hemophilia, development of antibodiesto blood coagulation factors, (3) liver disease, (4) bleeding insituations where pressure to the bleeding area is ineffective or cannotbe applied, such as bleeding from diffuse areas of injury, bleeding fromesophageal varices, gastritis, cystitis, endometritis, vascular injuryor head trauma, (5) bleeding in situations where platelets are notpresent in adequate number, (6) bleeding in situations where plateletsare not functioning properly, or (7) as an adjunct to use of recombinantFactor VIIa (an FDA-approved drug) in the treatment of patients withbleeding disorders.

Use in Inducing Thrombosis

Also envisioned is the use of sTF-annV or similar chimeric proteinsdescribed herein to induce thrombosis in blood vessels not related to acancerous state, but which are potentially deleterious to the host. Suchcollections of blood vessels include, but are not limited to,telangiectasias, arterial malformations, venous malformations, capillarymalformations, lymphatic malformations, arterio-venous malformations,hemangiomas, or aneurysms. In addition, the invention envisions usingthe described proteins to treat areas characterized by the growth ofstructurally normal blood vessels in a manner or site that maycompromise the well being of the host, sometimes collectively referredto as areas of “neovascularization”. It is envisioned that sTF-annV orvariations thereof would be administered to the host (by any of thevariety of routes described herein) in order to induce localizedthrombosis in a therapeutic manner. It may be necessary to utilizeanother treatment modality before, or simultaneously, in order toimprove the efficacy of sTF-annV. Such adjunctive treatments mightinclude drugs, chemicals, electrical stimulation, or radiation therapy.

Use in Cancer Treatment.

Annexin V binds to tumors and/or blood vessel cells within a tumor.Localization of sTF to tumors via annexin V (or other PS bindingstructure described herein) will lead to the initiation of bloodcoagulation within the tumor, thereby cutting off the flow of nutrientscarried into the tumor, and leading to death of the tumor. The presentinvention utilizes the AnnV domain or of the PS binding structures tolocalize the chimeric protein to the tumor.

Use in Targeting Therapeutic Moieties.

The invention also contemplates the use of sTF-annV to delivertherapeutic materials, such as radioactive materials or chemotherapeuticcompounds to tumors or abnormal vascular beds. In one embodiment forexample, the therapeutically augmented (for example, radioactive)sTF-annV is injected into a cancer-bearing host. Localization of thetherapeutically active sTF-annexinV into the tumor vessels, perhapsfollowing the administration of chemotherapy, radiotherapy, or othermanipulations designed to increase the targeting of the sTF-annV to thedesired region, will augment the therapeutic effect of the molecule. Theuse of such modified sTF-annV molecules linked to a radioactive orchemotherapeutic moiety will be of use in treating any of a variety ofcancers, vascular tumors, or vascular malformations. Examples of howchemotherapeutic or radioactive materials can be linked to proteins areshown in U.S. Pat. Nos. 6,074,643; 6,696,478; 6,403,771; 5,620,675;5,346,686; 5,219,556; 6,852,318, 5,863,538; 7,001,991; 5,108,987;5,000,935; 4,895,714; and 4,886,780, each of which is hereby expresslyincorporated herein by reference.

A “therapeutically effective amount” or “biologically-effective amount”of a compound of the present invention refers to an amount which iseffective in controlling, inhibiting, reducing, or promoting hemostasisor thrombosis or controlling, inhibiting, or reducing tumor growth. Theterm “controlling” is intended to refer to all processes wherein theremay be a slowing, interrupting, arresting, or stopping of theprogression of the disease or condition and does not necessarilyindicate a total elimination of all disease symptoms.

The term “therapeutically effective amount” or “biologically effectiveamount” is further meant to define an amount resulting in theimprovement of any parameters or clinical symptoms characteristic of thedisease condition. The actual dose will be different for the variousspecific molecules, and will vary with the patient's overall condition,the seriousness of the symptoms, and counter indications.

As used herein, the term “subject” or “patient” refers to a warm bloodedanimal such as a mammal which is afflicted with a particular diseasestate and needing the therapy as described herein. It is understood thatguinea pigs, dogs, cats, rats, mice, horses, cattle, sheep, 200 animals,livestock, and humans are examples of animals within the scope of themeaning of the term.

A therapeutically effective amount or biologically-effective amount ofthe compound used in the treatment described herein can be readilydetermined by the attending diagnostician, as one skilled in the art, bythe use of conventional techniques and by observing results obtainedunder analogous circumstances. In determining the therapeuticallyeffective dose, a number of factors are considered by the attendingdiagnostician, including, but not limited to: the species of mammal; itssize, age, and general health; the specific disease or conditioninvolved; the degree of or involvement or the severity of the disease orcondition; the response of the individual subject; the particularcompound administered; the mode of administration; the bioavailabilitycharacteristic of the preparation administered; the dose regimenselected; the use of concomitant medication; and other relevantcircumstances.

A therapeutically effective amount or biologically effective amount of acompound of the present invention also refers to an amount of thecompound which is effective in treating the disease condition or anothercondition described herein.

A therapeutically effective amount or biologically effective amount ofthe compositions of the present invention will generally containsufficient active ingredient (i.e., the chimeric protein) to deliverfrom about 0.1 ng/kg to about 100 mg/kg (weight of activeingredient/body weight of patient). Preferably, the composition willdeliver at least 10 ng/kg to 50 mg/kg, and more preferably at least 100ng/kg to 10 mg/kg.

Practice of the method of the present invention comprises administeringto a subject a therapeutically effective amount or biologicallyeffective amount of the active ingredient, in any suitable systemic orlocal formulation, in an amount effective to deliver the dosages listedabove. The dosage can be administered on a one-time basis, or (forexample) from one to five times per day or once or twice per week, orcontinuously via a venous drip, depending on the desired therapeuticeffect.

As noted, preferred amounts and modes of administration are able to bedetermined by one skilled in the art. One skilled in the art ofpreparing formulations can readily select the proper form and mode ofadministration depending upon the particular characteristics of thecompound selected, the disease state or condition to be treated, thestage of the disease or condition, and other relevant circumstancesusing formulation technology known in the art, described, for example,in Remington's Pharmaceutical Sciences, latest edition, Mack PublishingCo.

Pharmaceutical compositions can be manufactured utilizing techniquesknown in the art. Typically the therapeutically effective amount of thecompound will be admixed with a pharmaceutically acceptable carrier.

The compounds or compositions of the present invention may beadministered by a variety of routes, for example, orally or parenterally(i.e., subcutaneously, intravenously, intramuscularly,intraperitoneally, or intratracheally), intraocularly, orintracranially.

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, lozenges, melts,powders, suspensions, or emulsions. Solid unit dosage forms can becapsules of the ordinary gelatin type containing, for example,surfactants, lubricants and inert fillers such as lactose, sucrose, andcornstarch or they can be sustained release preparations. The dosagesmay be enterically coated.

In another embodiment, the compounds of this invention can be tablettedwith conventional tablet bases such as lactose, sucrose, and cornstarchin combination with binders, such as acacia, cornstarch, or gelatin,disintegrating agents such as potato starch or alginic acid, and alubricant such as stearic acid or magnesium stearate. Liquidpreparations are prepared by dissolving the active ingredient in anaqueous or non-aqueous pharmaceutically acceptable solvent which mayalso contain suspending agents, sweetening agents, flavoring agents, andpreservative agents as are known in the art.

For parenteral administration, the compounds may be dissolved in aphysiologically acceptable pharmaceutical carrier and administered aseither a solution or a suspension. Illustrative of suitablepharmaceutical carriers are water, saline, dextrose solutions, fructosesolutions, ethanol, or oils of animal, vegetative, or synthetic origin.The pharmaceutical carrier may also contain preservatives, and buffersas are known in the art.

The compounds of this invention can also be administered topically undercertain conditions. This can be accomplished by simply preparing asolution of the compound to be administered, preferably using a solventknown to promote transdermal absorption such as ethanol or dimethylsulfoxide (DMSO) with or without other excipients. Preferably topicaladministration will be accomplished using a patch either of thereservoir and porous membrane type or of a solid matrix variety.

As noted above, the compositions can also include an appropriatecarrier. For topical use, any of the conventional excipients may beadded to formulate the active ingredients into a lotion, ointment,powder, cream, spray, or aerosol. For surgical implantation, the activeingredients may be combined with any of the well-known biodegradable andbioerodible carriers, such as polylactic acid and collagen formulations.Such materials may be in the form of solid implants, sutures, sponges,wound dressings, and the like. In any event, for local use of thematerials, the active ingredients usually be present in the carrier orexcipient in a weight ratio of from about 1:1000 to 1:20,000, but arenot limited to ratios within this range. Preparation of compositions forlocal use are detailed in Remington's Pharmaceutical Sciences, latestedition, (Mack Publishing).

Additional pharmaceutical methods may be employed to control theduration of action. Increased half-life and controlled releasepreparations may be achieved through the use of polymers to conjugate,complex with, or absorb the protein described herein. The controlleddelivery and/or increased half-life may be achieved by selectingappropriate macromolecules (for example, polysaccharides, polyesters,polyamino acids, homopolymers polyvinyl pyrrolidone,ethylenevinylacetate, methylcellulose, or carboxymethylcellulose, andacrylamides such as N-(2-hydroxypropyl) methacrylamide, and theappropriate concentration of macromolecules as well as the methods ofincorporation, in order to control release.

Another possible method useful in controlling the duration of action bycontrolled release preparations and half-life is incorporation of thechimeric protein or its functional derivatives into particles of apolymeric material such as polyesters, polyamides, polyamino acids,hydrogels, poly(lactic acid), ethylene vinylacetate copolymers,copolymer micelles of, for example, PEG and poly(l-aspartamide).

The half-life of the proteins described herein can be extended by theirbeing conjugated to other molecules such as polymers using methods knownin the art to form drug-polymer conjugates. For example, the proteinscan be bound to molecules of inert polymers known in the art, such as amolecule of polyethylene glycol (PEG) in a method known as “pegylation”.Pegylation can therefore extend the in vivo lifetime and thustherapeutic effectiveness of the protein molecule. Pegylation alsoreduces the potential antigenicity of the protein molecule. Pegylationcan also enhance the solubility of the proteins thereby improving theirtherapeutic effect. PEGs used may be linear or branched-chain.

PEG molecules can be modified by functional groups, for example as shownin Harris et al., (9) the entirety of which is hereby expreslyincorporated herein by reference, and the amino terminal end of theprotein, or cysteine residue if present, or other linking amino acidtherein can be linked thereto, wherein the PEG molecule can carry one ora plurality of one or more types of the proteins or, the protein cancarry more than one PEG molecule.

By “pegylated ptotein” is meant a protein of the present inventionhaving a polyethylene glycol (PEG) moiety covalently bound to an aminoacid residue or linking group of the protein.

By “polyethylene glycol” or “PEG” is meant a polyalkylene glycolcompound or a derivative thereof, with or without coupling agents orderviatization with coupling or activating moeities (e.g., with thiol,triflate, tresylate, azirdine, oxirane, or preferably with a maleimidemoiety). Compounds such as maleimido monomethoxy PEG are exemplary oractivated PEG compounds of the invention. Other polyalkylene glycolcompounds, such as polypropylene glycol, may be used in the presentinvention. Other appropriate polymer conjugates include, but are notlimited to, non-polypeptide polymers, charged or neutral polymers of thefollowing types: dextran, colominic acids or other carbohydrate basedpolymers, biotin deriviatives and dendrimers, for example. The term PEGis also meant to include other polymers of the class polyalkyleneoxides.

The PEG can be linked to any N-terminal amino acid of the protein,and/or can be linked to an amino acid residue downstream of theN-terminal amino acid, such as lysine, histidine, tryptophan, asparticacid, glutamic acid, and cysteine, for example or other such amino acidsknown to those of skill in the art. Cysteine-pegylated proteins, forexample, are created by attaching polyethylene glycol to a thio group ona cysteine residue of the protein.

The chemically modified chimeric proteins contain at least one PEGmoiety, preferably at least two PEG moieties, up to a maximum number ofPEG moieties bound to the protein without abolishing activity, e.g., thePEG moiety(ies) are bound to an amino acid residue preferably at or nearthe N-terminal portion of the protein.

The PEG moiety attached to the protein may range in molecular weightfrom about 200 to 20,000 MW. Preferably the PEG moiety will be fromabout 1,000 to 8,000 MW, more preferably from about 3,250 to 5,000 MW,most preferably about 5,000 MW.

The actual number of PEG molecules covalently bound per chemicallymodified protein of the invention may vary widely depending upon thedesired protein stability (i.e. serum half-life).

Proteins contemplated herein can be linked to PEG molecules usingtechniques shown, for example (but not limited to), in U.S. Pat. Nos.,4,179,337; 5,382,657; 5,972,885; 6,177,087; 6,165,509; 5,766,897; and6,217,869; the specifications and drawings each of which are herebyexpressly incorporated herein by reference. Alternatively, the serumhalf-life of the chimeric protein can be extended by lengthening thechimeric proteins with additional amino acids or polypeptides, forexample by having several sTF domains in a series, or by attaching asuitable protein which is compatible with the subject being treated,such as human serum albumin in human subjects.

Alternatively, it is possible to entrap the chimeric proteins inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatine-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules), or in macroemulsions. Such techniques are disclosed inthe latest edition of Remington's

Pharmaceutical Sciences.

U.S. Pat. No. 4,789,734 describe methods for encapsulating biochemicalsin liposomes and is hereby expressly incorporated by reference herein.Essentially, the material is dissolved in an aqueous solution, theappropriate phospholipids and lipids added, along with surfactants ifrequired, and the material dialyzed or sonicated, as necessary. A reviewof known methods is by G. Gregoriadis (10). Microspheres formed ofpolymers or proteins are well known to those skilled in the art, and canbe tailored for passage through the gastrointestinal tract directly intothe blood stream. Alternatively, the agents can be incorporated and themicrospheres, or composite of microspheres, implanted for slow releaseover a period of time, ranging from days to months. See, for example,U.S. Pat. Nos. 4,906,474; 4,925,673; and 3,625,214 which areincorporated by reference herein.

When the composition is to be used as an injectable material, it can beformulated into a conventional injectable carrier. Suitable carriersinclude biocompatible and pharmaceutically acceptable phosphate bufferedsaline solutions, which are preferably isotonic.

For reconstitution of a lyophilized product in accordance with thisinvention, one may employ a sterile diluent, which may contain materialsgenerally recognized for approximating physiological conditions and/oras required by governmental regulation. In this respect, the sterilediluent may contain a buffering agent to obtain a physiologicallyacceptable pH, such as sodium chloride, saline, phosphate-bufferedsaline, and/or other substances which are physiologically acceptableand/or safe for use. In general, the material for intravenous injectionin humans should conform to regulations established by the Food and DrugAdministration, which are available to those in the field.

The pharmaceutical composition may also be in the form of an aqueoussolution containing many of the same substances as described above forthe reconstitution of a lyophilized product.

The compounds can also be administered as a pharmaceutically acceptableacid- or base-addition salt, formed by reaction with inorganic acidssuch as hydrochloric acid, hydrobromic acid, perchloric acid, nitricacid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organicacids such as formic acid, acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,maleic acid, and fumaric acid, or by reaction with an inorganic basesuch as sodium hydroxide, ammonium hydroxide, potassium hydroxide, andorganic bases such as mono-, di-, trialkyl and aryl amines andsubstituted ethanolamines.

As mentioned above, the compounds of the invention may be incorporatedinto pharmaceutical preparations which may be used for therapeuticpurposes. However, the term “pharmaceutical preparation” is intended ina broader sense herein to include preparations containing a chimericprotien composition in accordance with this invention, used not only fortherapeutic purposes but also for reagent or diagnostic purposes asknown in the art, or for tissue culture. The pharmaceutical preparationintended for therapeutic use should contain a “pharmaceuticallyacceptable” or “therapeutically effective amount” of a chimeric protein,i.e., that amount necessary for preventative or curative healthmeasures. If the pharmaceutical preparation is to be employed as areagent or diagnostic, then it should contain reagent or diagnosticamounts of a chimeric protein.

The present invention is not to be limited in scope by the specificembodiments described herein, since such embodiments are intended as butsingle illustrations of one aspect of the invention and any functionallyequivalent embodiments are within the scope of this invention. Indeed,various modifications of the compounds and methods of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description.

Each of the references, patents or publications cited herein isexpressly incorporated herein by reference in its entirety.

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1. A method of inducing hemostasis or thrombosis in an animal,comprising: providing a chimeric protein comprising a phosphatidylserinebinding domain and a soluble tissue factor domain comprising SEQ ID NO:2or a mutant thereof having human Tissue Factor activity; andadministering the chimeric protein to the animal in an amount effectiveto promote coagulation in a vascularized area of the animal.
 2. Themethod of claim 1 wherein the vascularized area of the animal isnon-cancerous.
 3. The method of claim 1 wherein the phosphatidylserinebinding domain comprises an Annexin.
 4. The method of claim 1 whereinthe phosphatidylserine binding domain comprises human Annexin V.
 5. Themethod of claim 1 wherein the chimeric protein mutant of SEQ ID NO:2comprises at least one substitution selected from the group consistingof ala at position 13, 131, 163, 164, or 183, asn at position 42, or138, trp at position 48, ser at position 52, asp at position 128, gin atposition 129, 163, or 164, and glu at position 163 or
 164. 6. The methodof claim 1 wherein the chimeric protein further comprises ser-gly or glyat the amino-terminus of SEQ ID NO:2.
 7. The method of claim 1 whereinthe chimeric protein further comprises a linker connecting the solubletissue factor domain and the phosphatidylserine binding domain.
 8. Themethod of claim 1 wherein the vascularized area of the animal isbleeding due to presence of an anticoagulant drug; a lack of acoagulation factor due to trauma, transfusion, antibodies, or congenitalconditions; liver disease; vascular or head injury; gastrointestinalconditions including gastritis, ulcer, and esophageal varices; cystitis,endometritis; or bleeding due to insufficient platelets or improperlyfunctioning platelets.
 9. The method of claim 1 further comprisingadministering the chimeric protein with recombinant Factor VIIa.
 10. Themethod of claim 1 wherein the vascularized area comprises atelangiectasia, arterial malformation, venous malformation, capillarymalformation, lymphatic malformation, arterio-venous malformation,hemangioma, or aneurysm.
 11. The method of claim 1 wherein the chimericprotein is administered locally in order to induce a local thrombosis.12. The method of claim 1 further comprising administering a drug,chemical, electrical stimulus or radiation in adjunct to the step ofadministering the chimeric protein.
 13. The method of claim 1 whereinthe chimeric protein further comprises a therapeutic compound ormaterial wherein the chimeric protein serves to deliver the therapeuticcompound or material to a site in need of treatment therewith.
 14. Themethod of claim 13 wherein the vascularized area to which thetherapeutic compound or material is delivered is a tumor or abnormalvascular bed.
 15. The method of claim 13 wherein the therapeuticmaterial or compound is chemotherapeutic or radioactive.